This application addresses broad Challenge Area (03) Biomarker Discovery and Validation and specific Challenge Topic, 03-CA-103 Reagents for rapid screening of human tumor cells for defects in DNA repair and/or replication. Defective DNA repair is common in tumors, and loss-of-function alleles of genes involved in double strand break (DSB) repair can cause hereditary cancer predisposition syndromes. DSBs are the most dangerous form of DNA damage, since their misrepair can cause gross chromosomal arrangements and promote cancer. DSBs are repaired by one of two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The recent development of poly(ADP ribose) polymerase (PARP) inhibitors to sensitize tumor cells defective for HR (such as those lacking BRCA1 or BRCA2) has highlighted the therapeutic synergy inherent in targeting repair pathways other than HR in these cells. We believe that PARP inhibitors are not the only class of reagent that will show such synergy. We propose that NHEJ inhibition in cells defective for HR may represent an additional target for cancer therapy. To this end, we have developed a novel, rapid and highly sensitive assay for NHEJ in mammalian cells. We proposed to use this and existing HR assays, developed by us, for the following three Aims: Aim 1. By conducting a siRNA screen for genes that promote NHEJ, to identify new genes that regulate mammalian NHEJ. Aim 2. By conducting a chemical screen for compounds that inhibit mammalian NHEJ, to identify new therapeutics that inhibit mammalian NHEJ. Aim 3. To develop a rapid, sensitive and quantitative assay for screening of human tumor cells for defects in homologous recombination. PUBLIC HEALTH RELEVANCE: Defects in double strand break (DSB) repair are common in human cancers, and offer an exciting potential new target for therapy of human cancer. New tools developed in our laboratory will allow us to rapidly screen the human genome for new genes that regulate DSB repair. We will use the same technology to identify new molecules that target DSB repair for therapy, and to rapidly assess human cancers for defective DSB repair.